Recently, 2.3 million women were diagnosed with BC globally [16]. When the BC is identified early treatment can be highly effective. This treatment consists of a combination of surgical, radiation therapy, and medication (chemotherapy, hormonal therapy, and targeted biological therapy), prevent cancer growth and spread, thereby saving lives. Therefore, studies for advances in the treatment of this type of cancer have priority.
Radiotherapy is used to treat effectively various cancers in different stages, included advanced. However, has been shown that patients can acquisition of radioresistance during radiotherapy, which causes therapy to become ineffective [17]. Apart from this, damage to normal tissues is unavoidable during high-dose radiation treatments. For the radiotherapy treatment to be considered successful it is necessary that an increase in tumor cell death occurs while the adverse effects in the surroundings of healthy tissues are minimized. Thus, understanding the mechanisms that enhance radioresistance is important for advancing the development of new radiotherapeutic strategies [18].
High doses mainly cause damage to cellular DNA and proteins [19, 20], while doses of approximately 200 mGy can have anti-inflammatory events. However, it is not possible to know all inflammatory factors and their signaling pathways for all types of radiation doses administered. Distinguishing between effective doses with less damage to surrounding tissues in the field of radiotherapy is challenging. Our results, where different cell models received a single dose of 2 Gy, show that there was no change in the cellular microenvironment leading to changes in the mesenchymal epithelium.
The occurrence of DSB in the DNA molecule is considered the most critical damage and can lead to cell death, mutations, and genomic rearrangements that contribute to the development of cancer [21]. The activation of histone H2AX (γH2AX), important signaling damage in the DNA molecule, was investigated. [22], using the biomarker of histone H2AX phosphorylation on serine 139 (γH2AX), which is considered a sensitive and accurate marker of IR-induced DSBs [23]. The MCF-10A non-tumor cell had a greater number of γH2AX foci 15 min after irradiation, while in the MCF-7 tumor cell the greatest number of foci occurs at 30 min after irradiation. However, at 60 min the number of foci of the two strains is declining. In the MDA-MB-231 tumor cell, the number of foci increases over the time of experimental analysis. This suggests inefficient repair of that cell. It is also observed that the repair response in tumorigenic cells is slower when compared to the non-tumor cell MCF-10 A, which is successful in repair after 15 min. This data suggests that these cells have a repair mechanism sufficient to repair the damage to the DNA molecule [24]. Even so, it is not possible to say that all the damages were repaired efficiently, because no analysis of the repair mechanisms was carried out. This temporal reduction in the number of cells labeled for γH2AX may be associated with repair events of DNA breaks, and the permanence of others with the label may be associated with an incorrect repair. This is in agreement with the literature that suggests that tumor cells may have lower efficiency in repair mechanisms [25]. The persistence of γH2AX foci in DNA is associated with failures in the repair mechanism that can lead to genomic instability, cell death, and carcinogenesis [26, 27].
The results obtained by immunofluorescence analysis showed differences between the sizes and quantity of γH2AX foci, being specific for each cell line. Both MDA-MB-231 and MCF-7 tumor lines exhibited fewer γH2AX foci when compared to the non-tumor cell MCF-10A. Furthermore, these quantification results suggest that the MCF-10A non-tumor cell is more radiosensitive while the MDA-MB-231 tumor cell is more radioresistant when irradiated with 2 Gy.
In this research, it was demonstrated that the evaluated strains had their ability to form colonies inhibited with the dose of 2 Gy. However, there are distinctions in the proliferative capacity of cells. Tumor cell lines had a higher rate of apoptosis 24 h after being irradiated, that is, IR was able to induce damage to DNA molecules, which could not be repaired by the cell's DNA system. However, the triple-negative adenocarcinoma cell, MDA-MB-231, when compared to the MCF-7 cell, has a higher survival fraction 14 days after receiving the 2 Gy dose, showing to be more aggressive in terms of proliferation. The non-tumor cell MCF-10A has a higher rate of apoptosis 48 h after irradiation. However, its clonogenic capacity suggests that the radiation dose used was not able to promote sufficient cellular alterations that lead a normal cell to undergo some type of malignant transformation. For, the transformation of non-tumor cells into neoplastic cells is a complex event that develops in multiple stages [14, 28, 29]. The ability to deal with DNA damage is highly dependent on the cell type, and this defines its radiosensitivity. Cell death is the loss of proliferative capacity, and survival is related to clonogenic capacity.
In addition to γH2AX labeling, apoptosis, and survival, this research aimed to evaluate characteristics directly related to the carcinogenesis process, such as EMT. Some studies demonstrate that EMT has been observed in breast cells after irradiation in radiotherapy [30, 31]. Cells with an epithelial phenotype acquire a mesenchymal phenotype. And this may be associated with disease recurrence and radioresistance. Therefore, we evaluated EMT markers when cells, MCF-10A, MCF-7, and MDA-MB-231, received a single dose of 2 Gy.
It was observed that the epithelial marker E-cadherin was expressed positively in the normal MCF-10A cell, and in tumor cells it was expressed negatively in the MCF-7 cell, showing that this cell lost adherence [30, 31, 32, 33], and had MMP9 degradation, which can facilitate the process of invasion and metastasis [31, 32]. However, this cell did not show changes in the mesenchymal markers N-cadherin, Vimentin, and Snail, while in the MDA-MB-231 cell this marker is absent. The absence of E-cadherin indicates a lack of adherence and increased motility, ability to migrate, invade and metastasize. However, after irradiation with a single dose of 2 Gy, there was no change in the mesenchymal markers N-cadherin, Vimentin, and Snail. This cell had its invasiveness doubled 72 h after irradiation, showing the absence of E-cadherin. Because it invaded and the ability to carry out metastasis is known, the transendothelial migration of this cell was evaluated. It can be said that 72 h after receiving a dose of 2 Gy, the MDA-MB-231 cell is induced to invade, but it cannot migrate through the endothelial cell monolayer. These data are in agreement with Young et al [34]. Young irradiated with 2.3 Gy the cell lines MCF-7 and MDA-MB-231 and observed that the marker vimentin did not show variation in expression. Furthermore, this does increase MDA-MB-231 invasion but did not increase invasion through the reconstituted basement membrane. It is important to note that these results were observed at the clinically used dose for standard fractionated radiotherapy treatment for CM.